scholarly journals CRIB effector disorder: exquisite function from chaos

2018 ◽  
Vol 46 (5) ◽  
pp. 1289-1302 ◽  
Author(s):  
Darerca Owen ◽  
Helen R. Mott
Keyword(s):  
Protein Binding ◽  
G Protein ◽  
Multiple Roles ◽  
Cellular Processes ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Binding Regions ◽  
Protein Binding Region ◽  
Basic Region ◽  
Liquid Liquid Phase Separation

The CRIB (Cdc42/Rac interactive binding) family of small G-protein effectors contain significant regions with intrinsic disorder. The G-protein-binding regions are contained within these intrinsically disordered regions. Most CRIB proteins also contain stretches of basic residues associated with their G-protein-binding regions. The basic region (BR) and G-protein-binding region together allow the CRIB effectors to bind to their cognate G-protein via a dock- and coalesce-binding mechanism. The BRs of these proteins take on multiple roles: steering G-protein binding, interacting with elements of the membrane and regulating intramolecular regulatory interactions. The ability of these regions of the CRIBs to undergo multivalent interactions and mediate charge neutralizations equips them with all the properties required to drive liquid–liquid phase separation and therefore to initiate and drive signalosome formation. It is only recently that the structural plasticity in these proteins is being appreciated as the driving force for these vital cellular processes.

scholarly journals Transcription Regulators and Membraneless Organelles Challenges to Investigate Them

2021 ◽  
Vol 22 (23) ◽  
pp. 12758
Author(s):  
Katarzyna Sołtys ◽  
Andrzej Ożyhar
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Cellular Processes ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Experimental Systems ◽  
Transcription Regulators ◽  
Crucial Information ◽  
Liquid Liquid Phase Separation

Eukaryotic cells are composed of different bio-macromolecules that are divided into compartments called organelles providing optimal microenvironments for many cellular processes. A specific type of organelles is membraneless organelles. They are formed via a process called liquid–liquid phase separation that is driven by weak multivalent interactions between particular bio-macromolecules. In this review, we gather crucial information regarding different classes of transcription regulators with the propensity to undergo liquid–liquid phase separation and stress the role of intrinsically disordered regions in this phenomenon. We also discuss recently developed experimental systems for studying formation and properties of membraneless organelles.

scholarly journals The arrested state of processing bodies supports mRNA regulation in early development

2021 ◽  
Author(s):  
M. Sankaranarayanan ◽  
Ryan J. Emenecker ◽  
Marcus Jahnel ◽  
Irmela R. E. A. Trussina ◽  
Matt Wayland ◽  
...  
Keyword(s):  
Physical Properties ◽  
Electrostatic Interactions ◽  
Processing Bodies ◽  
P Bodies ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Dead Box ◽  
Premature Release ◽  
Liquid Liquid Phase Separation

ABSTRACTBiomolecular condensates that form via liquid-liquid phase separation can exhibit diverse physical states. Despite considerable progress, the relevance of condensate physical states forin vivobiological function remains limited. Here, we investigated the physical properties ofin vivoprocessing bodies (P bodies) and their impact on mRNA storage in matureDrosophilaoocytes. We show that the conserved DEAD-box RNA helicase Me31B forms P body condensates which adopt a less dynamic, arrested physical state. We demonstrate that structurally distinct proteins and hydrophobic and electrostatic interactions, together with RNA and intrinsically disordered regions, regulate the physical properties of P bodies. Finally, using live imaging, we show that the arrested state of P bodies is required to prevent the premature release ofbicoid(bcd) mRNA, a body axis determinant, and that P body dissolution leads tobcdrelease. Together, this work establishes a role for arrested states of biomolecular condensates in regulating cellular function in a developing organism.

scholarly journals Overexpression of the microtubule-binding protein CLIP-170 induces a +TIP network superstructure consistent with a biomolecular condensate

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260401
Author(s):  
Yueh-Fu O. Wu ◽  
Annamarie T. Bryant ◽  
Nora T. Nelson ◽  
Alexander G. Madey ◽  
Gail F. Fernandes ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Fluorescence Recovery After Photobleaching ◽  
Liquid Droplet ◽  
Large Patch ◽  
Cellular Processes ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Structural Fluctuations ◽  
The Web ◽  
Disordered Regions

Proper regulation of microtubule (MT) dynamics is critical for cellular processes including cell division and intracellular transport. Plus-end tracking proteins (+TIPs) dynamically track growing MTs and play a key role in MT regulation. +TIPs participate in a complex web of intra- and inter- molecular interactions known as the +TIP network. Hypotheses addressing the purpose of +TIP:+TIP interactions include relieving +TIP autoinhibition and localizing MT regulators to growing MT ends. In addition, we have proposed that the web of +TIP:+TIP interactions has a physical purpose: creating a dynamic scaffold that constrains the structural fluctuations of the fragile MT tip and thus acts as a polymerization chaperone. Here we examine the possibility that this proposed scaffold is a biomolecular condensate (i.e., liquid droplet). Many animal +TIP network proteins are multivalent and have intrinsically disordered regions, features commonly found in biomolecular condensates. Moreover, previous studies have shown that overexpression of the +TIP CLIP-170 induces large “patch” structures containing CLIP-170 and other +TIPs; we hypothesized that these structures might be biomolecular condensates. To test this hypothesis, we used video microscopy, immunofluorescence staining, and Fluorescence Recovery After Photobleaching (FRAP). Our data show that the CLIP-170-induced patches have hallmarks indicative of a biomolecular condensate, one that contains +TIP proteins and excludes other known condensate markers. Moreover, bioinformatic studies demonstrate that the presence of intrinsically disordered regions is conserved in key +TIPs, implying that these regions are functionally significant. Together, these results indicate that the CLIP-170 induced patches in cells are phase-separated liquid condensates and raise the possibility that the endogenous +TIP network might form a liquid droplet at MT ends or other +TIP locations.

scholarly journals A new class of disordered elements controls DNA replication through initiator self-assembly

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Matthew W Parker ◽  
Maren Bell ◽  
Mustafa Mir ◽  
Jonchee A Kao ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Dna Replication ◽  
Self Assembly ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Linear Arrangement ◽  
New Class ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Liquid Liquid Phase Separation

The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

scholarly journals Combinatorial phosphorylation modulates the structure and function of the G protein γ subunit in yeast

2021 ◽  
Vol 14 (688) ◽  
pp. eabd2464
Author(s):  
Zahra Nassiri Toosi ◽  
Xinya Su ◽  
Ruth Austin ◽  
Shilpa Choudhury ◽  
Wei Li ◽  
...  
Keyword(s):  
G Protein ◽  
Posttranslational Modifications ◽  
Intrinsically Disordered Proteins ◽  
Structure And Function ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Gpcr Activation ◽  
Essential Components ◽  
And Function

Intrinsically disordered regions (IDRs) in proteins are often targets of combinatorial posttranslational modifications, which serve to regulate protein structure and function. Emerging evidence suggests that the N-terminal tails of G protein γ subunits, which are essential components of heterotrimeric G proteins, are intrinsically disordered, phosphorylation-dependent determinants of G protein signaling. Here, we found that the yeast Gγ subunit Ste18 underwent combinatorial, multisite phosphorylation events within its N-terminal IDR. G protein–coupled receptor (GPCR) activation and osmotic stress induced phosphorylation at Ser7, whereas glucose and acid stress induced phosphorylation at Ser3, which was a quantitative indicator of intracellular pH. Each site was phosphorylated by a distinct set of kinases, and phosphorylation of one site affected phosphorylation of the other, as determined through exposure to serial stimuli and through phosphosite mutagenesis. Last, we showed that phosphorylation resulted in changes in IDR structure and that different combinations of phosphorylation events modulated the activation rate and amplitude of the downstream mitogen-activated protein kinase Fus3. These data place Gγ subunits among intrinsically disordered proteins that undergo combinatorial posttranslational modifications that govern signaling pathway output.

scholarly journals Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation

2021 ◽  
Author(s):  
Tongyin Zheng ◽  
Carlos A. Castañeda
Keyword(s):  
Quality Control ◽  
Phase Separation ◽  
Liquid Phase ◽  
Protein Quality ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Uba Domain ◽  
Phase Separation Behavior ◽  
Separation Behavior ◽  
Liquid Liquid Phase Separation

AbstractShuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N-terminal ubiquitin-like (UBL) and C-terminal ubiquitin-associated (UBA) domains, respectively. Between these two folded domains are intrinsically disordered STI1-I and STI1-II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1-II region enables UBQLN2 to undergo liquid-liquid phase separation (LLPS) to form liquid dropletsin vitroand biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically-disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using NMR spectroscopy. To our surprise, aside from well-studied canonical UBL:UBA interactions, there also exist moderate and weak interactions between the UBL and STI1-I/STI1-II domains, and between the UBA domain and the linker connecting the two STI1 regions, respectively. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or STI1 domains on the phase behavior and physiological functions of UBQLN2.Impact of Work StatementZheng and Castañeda show that interplay between the folded domains and intrinsically disordered regions regulates liquid-liquid phase separation behavior of UBQLN2, a protein quality control (PQC) shuttle protein. Despite their similar size, the folded UBL and UBA domains inhibit and promote phase separation, respectively, due to their previously uncharacterized, asymmetric interactions with the middle intrinsically-disordered region. These results strongly suggest that PQC components, including proteasomal receptors, are likely to modulate UBQLN2 phase separation behavior in cells.

scholarly journals The (un)structural biology of biomolecular liquid-liquid phase separation using NMR spectroscopy

2020 ◽  
Vol 295 (8) ◽  
pp. 2375-2384 ◽  
Author(s):  
Anastasia C. Murthy ◽  
Nicolas L. Fawzi
Keyword(s):  
Phase Separation ◽  
Nmr Spectroscopy ◽  
Liquid Phase ◽  
Structural Biology ◽  
Liquid Phase Separation ◽  
Biological Processes ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Molecular Aspects ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids is a phenomenon that underlies membraneless compartmentalization of the cell. The underlying molecular interactions that underpin biomolecular LLPS have been of increased interest due to the importance of membraneless organelles in facilitating various biological processes and the disease association of several of the proteins that mediate LLPS. Proteins that are able to undergo LLPS often contain intrinsically disordered regions and remain dynamic in solution. Solution-state NMR spectroscopy has emerged as a leading structural technique to characterize protein LLPS due to the variety and specificity of information that can be obtained about intrinsically disordered sequences. This review discusses practical aspects of studying LLPS by NMR, summarizes recent work on the molecular aspects of LLPS of various protein systems, and discusses future opportunities for characterizing the molecular details of LLPS to modulate phase separation.

scholarly journals Overexpression of the microtubule-binding protein CLIP-170 induces a +TIP network superstructure consistent with a biomolecular condensate

2021 ◽  
Author(s):  
Yueh-Fu O. Wu ◽  
Annamarie T. Bryant ◽  
Nora T. Nelson ◽  
Alexander G. Madey ◽  
Gail F. Fernandes ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Fluorescence Recovery After Photobleaching ◽  
Liquid Droplet ◽  
Large Patch ◽  
Cellular Processes ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Structural Fluctuations ◽  
The Web ◽  
Disordered Regions

AbstractProper regulation of microtubule (MT) dynamics is critical for cellular processes including cell division and intracellular transport. Plus-end tracking proteins (+TIPs) dynamically track growing MTs and play a key role in MT regulation. +TIPs participate in a complex web of intra- and inter-molecular interactions known as the +TIP network. Hypotheses addressing the purpose of +TIP:+TIP interactions include relieving +TIP autoinhibition and localizing MT regulators to growing MT ends. In addition, we have proposed that the web of +TIP:+TIP interactions has a physical purpose, creating a superstructure that constrains the structural fluctuations of the fragile MT tip and thus acts as a polymerization chaperone. Many animal +TIP network proteins are multivalent and have intrinsically disordered regions, features commonly found in biomolecular condensates. This observation suggests that the +TIP network might under some conditions form a biomolecular condensate. Previous studies have shown that overexpression of the +TIP CLIP-170 induces large “patch” structures containing CLIP-170 and other +TIPs. To test the hypothesis that these patches might be biomolecular condensates, we used video microscopy, immunofluorescence staining, and Fluorescence Recovery After Photobleaching (FRAP). Our data show that the CLIP-170-induced patches have hallmarks indicative of a biomolecular condensate, one that contains +TIP proteins and excludes other known condensate markers. Moreover, bioinformatic studies demonstrate that the presence of intrinsically disordered regions is conserved in key +TIPs, implying that these regions are functionally significant. Together, these results indicate that the CLIP-170 induced patches in cells are phase-separated liquid condensates and raise the possibility that the endogenous +TIP network might form a liquid droplet at MT ends or other +TIP locations.

scholarly journals Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice

2021 ◽  
Vol 7 (1) ◽  
pp. eabd3568
Author(s):  
Nils Schneider ◽  
Franz-Georg Wieland ◽  
Deqiang Kong ◽  
Alexandra A. M. Fischer ◽  
Maximilian Hörner ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Phase Separation ◽  
Liquid Phase ◽  
Mammalian Cells ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Gene Switches ◽  
Liquid Liquid Phase Separation

Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.

scholarly journals A new class of disordered elements controls DNA replication through initiator self-assembly

2019 ◽  
Author(s):  
Matthew W. Parker ◽  
Maren Bell ◽  
Mustafa Mir ◽  
Jonchee A. Kao ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Dna Replication ◽  
Self Assembly ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Linear Arrangement ◽  
New Class ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Liquid Liquid Phase Separation

SUMMARYThe initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

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